Bonner zoologische Beiträge Band 55 (2006) Heft 3/4 Seiten 311–318 Bonn, November 2007 The cephalic sensory organs of Acteon tornatilis (Linnaeus, 1758) (Gastropoda Opisthobranchia) – cellular innervation patterns as a tool for homologisation* Sid STAUBACH & Annette KLUSSMANN-KOLB1) 1)Institute for Ecology, Evolution and Diversity – Phylogeny and Systematics, J. W. Goethe-University, Frankfurt am Main, Germany *Paper presented to the 2nd International Workshop on Opisthobranchia, ZFMK, Bonn, Germany, September 20th to 22nd, 2006 Abstract. Gastropoda are guided by several sensory organs in the head region, referred to as cephalic sensory organs (CSOs). This study investigates the CSO structure in the opisthobranch, Acteon tornatilis whereby the innervation pat- terns of these organs are described using macroscopic preparations and axonal tracing techniques. A bipartite cephalic shield and a lateral groove along the ventral side of the cephalic shield was found in A. tornatilis. Four cerebral nerves can be described innervating different CSOs: N1: lip, N2: anterior cephalic shield and lateral groove, N3 and Nclc: posterior cephalic shield. Cellular innervation patterns of the cerebral nerves show characteristic and con- stant cell clusters in the CNS for each nerve. We compare these innervation patterns of A. tornatilis with those described earlier for Haminoea hydatis (STAUBACH et al. in press). Previously established homologisation criteria are used in order to homologise cerebral nerves as well as the organs innervated by these nerves. Evolutionary implications of this homologisation are discussed. Keywords. Haminoea hydatis, Cephalaspidea, axonal tracing, homology, innervation patterns, lip organ, Hancock´s organ. 1. INTRODUCTION Gastropoda are guided by several organs in the head re- phylogenetic position of Acteonoidea within Opistho- gion which are assumed to have primarily chemo- and branchia unsettled. Acteonoidea are characterised by the mechanosensory functions (AUDESIRK 1979; DAVIS & presence of a prominent cephalic shield. This structure is MATERA 1982; BICKER et al.1982; EMERY 1992; CHASE also present in Cephalaspidea and has been considered to 2000; CROLL et al. 2003). In Opisthobranchia, these be an apomorphie of the Cephalaspidea (SC H M E K E L 1 9 8 5 ) . cephalic sensory organs (CSOs) present an assortment of H o w e v e r, the structure of the cephalic shields differs con- forms including rhinophores, labial tentacles, oral veils, siderably in Cephalaspidea and Acteonoidea with the lat- Hancock´s organs and cephalic shields. Recent investiga- ter possessing two distinct hemispheres while the cephal- tions of CSOs in Opisthobranchia have focussed prima- ic shield in the Cephalaspidea possesses uniform structure. rily on functional aspects (CROLL 1983; BOUDKO et al. Therefore, common origin of both types of cephalic 1999; CROLL et al. 2003) while homology of the differ- shields and thus homology is questionable. Further CSOs ent types of CSOs in different taxa has never been inves- have been described in Acteonoidea and Cephalaspidea tigated in detail. We want to clarify their homology in sep- such as lip organ and Hancock´s organ (RUDMAN 1971A; arate evolutionary lineages so as to elucidate key ques- RUDMAN 1971B; RUDMAN 1972a, b; RUDMAN 1972c; tions regarding character evolution and phylogeny. EDLINGER 1980). Acteon tornatilis belongs to the subgroup Acteonoidea, Since the presence of these organs in members of the formerly ascribed to the basal Cephalaspidea (ODHNER genus Acteon has been disputed by different authors 1939, BURN & THOMPSON 1998). However, recent inves- (EDLINGER 1980; SCHMEKEL 1985), absolute clarification tigations have either excluded the Acteonoidea from the is certainly necessary . The intention of this study is to de- Opisthobranchia (MIKKELSEN 1996) or proposed a sister scribe the structure emphasizing the innervation of the group relationship of Acteonoidea and the highly derived CSOs in the acteonid A. tornatilis. Our descriptions fo- Nudipleura (VONNEMANN et al. 2005) thus, rendering the cus on the cellular innervation patterns reconstructed for 312 Sid STAUBACH & Annette KLUSSMANN-KOLB: Cephalic sensory organ in Aceton the cerebral nerves using axonal tracing. In an earlier study ed on an objective slide dorsal side up in Entellan (VWR (STAUBACH et al. in press) these cellular innervation pat- International) and covered with a cover slip. Ten replicates terns were shown to be more adequate in homologising were prepared for each cerebral nerve of A. tornatilis. cerebral nerves than ganglionic origins of nerves (HUBER Samples with only a partial staining of the nerve were not 1993). By comparising the innervation patterns in A. tor - used because of possible incomplete innervation patterns. n a t i l i s to previously published data on H. hydatis Our criterion for a well-stained preparation was a dark blue (STA U B A C H et al. in pre s s), we want to survey whether the stained nerve indicating intact axons ( FR E D M A N 1 9 8 7 ). T h e preliminary characteristic cell clusters in the central nerv- Ni-Lys tracings were analysed by light microscopy (Le- ous system (CNS) of both taxa can be identified by ho- ica TCS 4D). Camera lucida drawings were digitalised fol- mologising cerebral nerves across taxa. Based on the ho- lowing the method of CO L E M A N ( 2 0 0 3 ) adapted for Corel- mologisation of the nerves innervating the CSOs we want DRAW 11. The somata in the innervation scemes occurs to clarify if A. tornatilis has homologous structures to the in all replicates. Somata only occurring in single samples CSOs of Cephalaspideans. It is our intent interest that we are not considered part of the schematics. The axonal path- shed light on the phylogenetic position and evolutionary ways are estimated over all replicates. Additionally, we history of Acteonoidea within the Opisthobranchia for fu- tested for asymmetries making axonal tracings (n = 2 to ture studies. 3) for each cerebral nerve of the left cerebral ganglion. 2.3. Scanning electron microscopy studies 2. MATERIALS AND METHODS The specimens were relaxed by an injection of 7 % Mg- 2.1. Specimens C l 2 in the foot. T h e r e a f t e r, the entire head region was dis- sected from the rest of the animal. The CSOs were fixed A. tornatilis (Fig. 1A) were collected in the wild at St. in 2,5 % glutaraldehyde, 1 % paraformaldehyde in 0,1M Michel en Greve (Brittany, France). They were then stored phosphate buffer (pH 7,2) at room temperature. For the alive at our lab in Frankfurt. Fourty specimens measur- SEM, the fixed CSOs were dehydrated through a graded ing a shell length between 15 and 20 mm were traced di- acetone series followed by critical point drying (CPD 030, rectly (5 to 15 days after collecting) and five were fixed BAL-TEC). Finally, they were spattered with gold (Sput- for SEM. ter-Coater, Agar Scientific) and examined with a Hitachi S4500 SEM. All photographs were taken using DISS (Dig- 2.2. Tracing studies ital Image Scanning System – Point Electronic) and sub- sequently adjusted for brightness and contrast with Corel Animals were relaxed with an injection of 7 % magne- PHOTO-PAINT 11. sium chloride. The central nervous system consisting of the cerebral, pleural and pedal ganglia was removed and placed in a small Petri dish containing filtered artificial 3. RESULTS seawater (ASW; Tropic Marin, Rebie-Bielefeld; GER- MANY). We then followed the procedures from CROLL 3.1. Organisation and innervation of the cephalic sen- & BAKER (1990) for Ni2+-lysine (Ni-Lys) tracing of ax- sory organs ons. Briefly, the nerves of the right cerebral ganglion were dissected free from the connective tissue. The nerves were A. tornatilis possesses a prominent bipartite cephalic shield cut and the distal tip was gently drawn into a glass mi- (cs) in which each hemisphere of this shield is divided in- cropipette using suction provided by an attached 2.5 ml to an anterior and a posterior lobe (Figs. 1A and B). Eyes syringe. Subsequently, the saline in the micropipette was are embedded deeply within the tissue of the shield. A l o n g replaced by a Ni-Lys solution (1.9g NiCl-6H2O, 3,5 g L- the lateral margin of the anterior lobe of the cephalic shield Lysine freebase in 20 ml double distilled H2O). The prepa- a groove is present (Fig. 1B, 2A). Hidden under the cs and ration was then incubated for 12–24 hours at 8º C to al- above the foot, the mouth opening is situated at the me- low transport of the tracer. The micropipette was then re- dian frontal edge (Fig. 2B) surrounded by the lip (not vis- moved and the ganglia were washed in ASW three times. ible in Figure 2B). We found four nerves innervating the The Ni-Lys was precipitated by the addition of five to ten CSOs (Fig.1B). The N1 (Nervus oralis) provides inner- drops of a saturated rubeanic acid solution in absolute Di- vation to the lip and small median parts of the anterior methylsulfoxide (DMSO). After 45 minutes the ganglia cephalic shield. The bifurcated N2 (Nervus labialis/labio- were transferred to 4 % paraformaldehyde (PFA) and fixed tentacularis) innervates the complete anterior cephalic for 4–12 hours at 4º C. Thereafter the ganglia were dehy- shield whereby the groove at the ventral anterior lobe of drated in an increasing ethanol series (70/80/90/99/99% the cephalic shield is especially innervated. The small N3 10 minutes each), cleared in methylsalicylate and mount- (Nervus tentacularis/rhinophoralis) innervates a little re- Bonner zoologische Beiträge 55 (2006) 313 gion of the posterior cephalic shield. The Nclc (Nervus For the N1 (n = 10) we identified six cerebral clusters clypei capitis) innervates the largest hind part of the pos- (Cnoc1-6) and one pedal cluster (Pdnoc1) in each sam- terior cephalic shield.